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Int. J. Life. Sci. Scienti. Res.

eISSN: 2455-1716
Hamid et al., 2018
DOI:10.21276/ijlssr.2018.4.5.9

Research Article

Phenotypic and Molecular Detection of Mycobacterium avium


subsp. paratuberculosis in Small Ruminants Clinically Suspected
with Johne᾿s Disease
1* 2 3 4
Mohamed A. I. Hamid , Galal Eldin E. Mohammed , Amel O. Bakhiet , Elhassan M. A. Saeed
1
Pathologist, Qassim University Teaching Veterinary Hospital, Saudi Arabia
2
Professor, Department of Microbiology, Pathology and Parasitology, Faculty of Veterinary Medicine, Sudan University of
Science and Technology, Sudan
3
Professor, Deanship of Scientific Research, Sudan University of Science and Technology, Sudan
4
Associate Professor, Department of Veterinary Medicine, College of Agriculture and Veterinary Medicine, Qassim
University, Saudi Arabia

*Address for Correspondence: Mr. Mohamed Alamin Ibrahim Hamid, Pathologist, Qassim University Teaching
Veterinary Hospital, Saudi Arabia
Received: 11 Apr 2018/ Revised: 09 Jun 2018/ Accepted: 16 Aug 2018

ABSTRACT
Johne's disease (JD) or Paratuberculosis (PTB) has gained a great attention by many industrial countries for its severing economic
losses and possibly zoonotic concerns. In the current study conventional clinical and direct microscopic examination compared to
real time polymerase chain reaction (RT-PCR) were used to diagnose JD in clinically suspected small ruminants. Clinical
examination revealed 130 (8.7%) suspected cases that showed history of emaciation and diarrhea out of the total examined
(1500) animals. Direct microscopy of Ziehl-Neelsen (ZN) stained smears (130) revealed 62 (47.7%) acid fast bacteria resembled
Mycobacterium avium subsp. paratuberculosis (MAP). RT-PCR insertion sequence gene (IS900) detected MAP in 25 (65.8%) out of
38 fecal samples harbored acid fast bacilli. We concluded and recommended that RT-PCR considers the most rapid confirmatory
method for screening and diagnosis of the MAP in comparison to low specific conventional phenotypic methods, which still
remained valuable techniques in the diagnosis of JD in developing countries.

Key-words: Johne's disease, Paratuberculosis, Acid fast bacteria, Ziehl-Neelsen stain, IS900 gene

INTRODUCTION
Johne's disease or Paratuberculosis is nowadays viewed hydrophobic cell wall, which is substantially thicker than
as one of the most serious chronic bacterial diseases of most other bacteria [2]. The thickness and fatty
ruminant which limits animal industry worldwide [1]. The composition of the cell wall render mycobacteria
disease is caused by Mycobacterium avium subsp. impermeable to hydrophilic nutrients and resist heavy
paratuberculosis (MAP) of the genus Mycobacterium. metals, disinfectants and antibiotics interaction [3].
MAP is an aerobic, non-motile acid fast bacterium. Recently MAP thought to be incriminated in Crohn’s
Members of the genus Mycobacterium have a lipid-rich, disease of humans. Clinical signs of Paratuberculosis in
small ruminants are not specific and could be confused
How to cite this article
with other diseases as intestinal parasitism, chronic
Hamid IMA, Mohammed GEE, Bakhiet AO, Ali Saeed EM.
Phenotypic and Molecular Detection of Mycobacterium avium malnutrition, ovine progressive pneumonia (OPP),
subsp. Paratuberculosis in Small Ruminants Clinically Suspected caseous lymphadenitis, environmental toxins, and cancer
with Johne᾿s Disease. Int. J. Life. Sci. Scienti. Res., 2018; 4(5): [4]
2019-2024. . Epidemiological data indicated the distribution of the
disease worldwide in both developed and developing
Access this article online countries in Europe, North America, South America, Asia,
www.ijlssr.com Australia and Africa [5]. In Saudi Arabia, in Grenada [6],
West Indies [7] and in Cyprian [8] many diagnostic tests
were used for diagnosis of paratuberculosis in ruminants.

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Int. J. Life. Sci. Scienti. Res. eISSN: 2455-1716
Hamid et al., 2018
DOI:10.21276/ijlssr.2018.4.5.9

Microscopic examination of Ziehl-Neelsen stain method GmbH, Mannheim-Germany) and 20 µl of proteinase k.


usually used, however, it has variable sensitivity and The mixture was mixed thoroughly and incubated for 10
specificity depending upon the stage of disease [4]. Fecal minutes at 65°C then after boiled for 10 minutes at 95°C,
culture demonstrates low sensitivity, require long centrifuged in low speed then chilled in ice from which
incubation period for growth as well as its labor intensive 400 µl were transferred to magna pure compact sample
so it cannot be recommended as a screening test to tube.
detect shedding of MAP in either goats or sheep [9].
DNA Purification- The extraction of DNA was carried out
Recently, IS900 and F57 insertion sequence genes have
in a fully automated Magna Pure Compact system (Roche
enabled the specific identification of minimum amounts
Diagnostics GmbH, Mannheim-Germany) according to
of bacterial DNA by different polymerase chain reaction
the manufacturer's instructions.
(PCR) techniques [10,11]. Therefore, the aim of this study
was to identify and confirm Mycobacterium avium subsp. Real Time Quantitative PCR- A real time qPCR assay, that
was applied for detection of presence of MAP bacteria,
MATERIALS AND METHODS
based on amplification of a 177 bp fragment of MAP
Clinical examination and sampling- A total number of
insertion element IS900 with set of specific primers and
1500 small ruminants were investigated in El Qassim
probe labeled with light cycler red 640 dye as described
region KSA. Based on history and clinical examination
by Beumer et. al. [13] and Rajeev et. al. [14]. In this assay,
animals showed signs of emaciation, diarrhea or
the Light cycler Fast Start DNA master hybprobe kit
softened feces, pasty stools, low-grade fever, lethargy,
(Roche Diagnostics GmbH, Mannheim-Germany) and the
and depression and chronic history of weight loss were
light mix Mycobacterium avium sp. paratuberculosis
supposed to be infected [4]. One hundred and thirty 130
(MAP) kit (TIB MOLBIOL GmbH-Berlin -Germany) was
clinical PTB suspected animals were selected and
used in an amplification reaction mixture. The mixture
subjected for microscopic screening test using fecal ZN
consisted of 2 µl 10x master mix, 2.4 µl 25 mM Mg2+, 2 µl
stained smears. Fecal samples were collected in sterile
of specific primers and probe sets solution and 5 µl DNA
containers from clinically suspected sheep and goats and
templates and completed to 20 µl with 8.4 µl PCR grade
preserved for further examination. The study proceeds
water. The PCR experiment was carried out in the Light
from February - October 2015 in Department of Clinical
Cycler 2.0 (Roche Diagnostics GmbH, Mannheim-
laboratory, Teaching Hospital, AL Qassim region, Saudi
Germany) with a protocol consisted of four thermal
Arabia.
program steps: initial denaturation one cycle at 95°C for
Traditional microscopic examination- From the collected 10min, amplification in 50 cycles, each cycle segmented
fecal samples direct smears were prepared, stained and to 95°C for 5sec, 62°C for 5sec, and 72°C for 15 sec and
microscopically examined for expected acid fast bacilli finally melting in one cycle with 3 thermal steps (95°C for
using ZN stain method [12]. 20 sec, 40°C for 20 sec and 85°C). The amplification
crossing (CP) and melting (Tm) points were detected in
Molecular methods 640 channels.
DNA extraction from fecal samples Statistical Analysis- The analysis was performed as
Preparation of the samples- Fresh, moist 38 fecal described in light cycler instrument operator's manual,
samples of clinical JD suspected animals were aliquoted using the second derivative maximum method. The
in 2ml cryo tubes and immediately frozen and stored at obtained data were analyzed with quantification analysis
-80°C until used. The fecal samples were prepared for mode and the amplification signals were reported as
extraction by using two grams protocol of MAP crossing points (cycle's threshold) in channel 640. For
Extraction System (Tetracore, USA) according to the further identification, the melting curve analysis mode
manufacturer's instructions. was performed and specific melting points were
detected by the same channel.
Pre-treatment of the samples- In this step, 200 µl
resuspension of the sample was mixed with 180 µl of
magna pure bacterial lysis buffer (Roche Diagnostics
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Int. J. Life. Sci. Scienti. Res. eISSN: 2455-1716
Hamid et al., 2018
DOI:10.21276/ijlssr.2018.4.5.9

RESULTS samples (n= 38) using IS900 gene revealed positive


Clinical examination of targeted animals showed 130 (75 results of 25 (62.5%) samples. These results indicated
goats and 55 sheep) JD suspected animals out of 1500 high sensitivity of the molecular test in contrast to the
total examined. Suspected animals showed clinical signs conventional clinical and microscopic methods. Twenty
include chronic weight loss, non-curable diarrhea, five (25) fecal samples tested were consistently positive
emaciation terminated by death. Microscopic screening for MAP insertion element IS900 by real time
of Ziehl-Neelsen's stain for rectal scraping smears from quantitative PCR (62.5%) (Table 2) and (Fig. 3 & 4). The
suspected cases, shown that 62 (47.7%) out of 130 were resulted cycle thresholds (Ct) range from 15.99 to 36.01,
harboring acid fast bacteria (Fig. 1, 2). of the positive with a mean of 28.2712 and melting points (Tm) range
cases 41 (54.7%) out of the 75 were goats and 21 (38.2%) from 66.03 to 68.85, with a mean of 67.9556 and
out of 55 were sheep (Table 1). RT-PCR examined fecal 0.47178 standard deviations.

Table 1: Numbers of clinically examined, suspected and acid fast positive cases

Total examined Clinical suspected Positive fecal Zn


Animal species Percentage
animals cases stain test
goat 900 75 41 54.7

sheep 600 55 21 38.2

Small ruminants 1500 130 62 47.7

Fig. 1: Direct rectal scrapings smear from sheep Fig. 2: Direct rectal scrapings smear from goat shows
shows acid fast clumps, Ziehl-Neelsen Stain X100 acid fast clumps, Ziehl-Neelsen Stain X100

Table 2: Quantitative RT-PCR, Ct and Tm results of PAM in the fecal samples in light cycler 2.0 and analyzed with
absolute and melting curve modes
No. of Samples CT * Tm**
1 29.11 68.32
2 27.98 68.18
3. 34.56 67.91
4. 26.89 68.05

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Int. J. Life. Sci. Scienti. Res. eISSN: 2455-1716
Hamid et al., 2018
DOI:10.21276/ijlssr.2018.4.5.9

5. 26.17 68.22
6. 25.02 68.31
7. 33.98 68.29
8. 28.96 68.09
9. 28.81 66.03
10. 28.25 67.92
11. 35.85 67.89
12. 30.81 67.95
13. 27.04 67.91
14. 33.56 67.86
15. 31.12 67.77
16. 28.48 67.98
17. 24.77 68.41
18. 28.69 67.85
19. 20.87 68.28
20. 25.25 68.00
21. 15.99 68.29
22. 27.03 68.08
23. 36.01 67.72
24. 15.99 68.29
25. 35.59 67.29
* Ct- Cycle's threshold, **Tm- Melting point temperature

Fig. 3: Displays the amplification curves and crossing points as analyzed by absolute analysis mode in light cycler 2.0

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Int. J. Life. Sci. Scienti. Res. eISSN: 2455-1716
Hamid et al., 2018
DOI:10.21276/ijlssr.2018.4.5.9

Fig. 4: Displays the melting curves & melting temp. points as analyzed by melting analysis mode in light cycler 2.0

DISCUSSION CONCLUSIONS
Detection of Paratuberculosis is important for control This study includes clinical screening of small ruminants
and eradication program. In current study the clinical for suspected cases of paratuberculosis, confirmed by
examination of targeted animals showed 130 JD both phenotypical and molecular identification were
suspected small ruminants out of 1500 total examined. able to document paratuberculosis among small
Initial microscopic screening of Ziehl-Neelsen's stain for ruminants in Qassim region. Based on the current
rectal scraping smears from suspected cases, showed finding, real time polymerase chain reaction is diagnostic
that 62 (47.7%) out of 130 were harboring acid fast and confirmed test of paratuberculosis among small
bacteria of the positive cases. These results were similar ruminants and a wide range of study among small
to studies conducted by Liapi, et al. [8] in Cyprian dairy ruminants is recommended for detection of the disease,
goat herds and it is higher than results obtained by Atif which could represent a potential zoonotic hazard of
et al. [6] and Kumthekar [7]. Although ZN staining is the infection among other animals in the region.
most rapid screening method, it lacks the required According to current finding we recommend the
sensitivity in the diagnosis of PTB; therefore, molecular application of RT-PCR as a confirmatory diagnostic test as
methods were alternatively used for rapid diagnosis of well as epidemiological screening test in combating and
MAP in farm animals [15]. Samples culture is labor eradication programs of paratuberculosis in farm
intensive and may require 8-24 weeks of incubation for animals.
colonies to be observed based on the type of media used
[16]
. RT-PCR examined fecal samples (n=38) using IS900 ACKNOWLEDGMENTS
gene revealed positive results of 25 (62.5%) samples. The authors would like to thank Dr. El Tigani, Asil A. El
These results indicated high sensitivity of the molecular Tigani for his kind participation in planning for the
test in contrast to the conventional clinical and research and collection of research samples.
microscopic methods. Twenty five (25) fecal samples
CONTRIBUTION OF AUTHORS
tested were consistently positive for MAP insertion
Research concept- Elhassan M. A. Saeed, Mohamed A. I.
element IS900 by real time quantitative PCR (62.5%).
Hamid
These results evaluated traditional methods (Zn stain) is
Research design- Mohamed A. I. Hamid, Amel O.
a lower specificity in compare to molecular methods
Bakhiet
(RT-PCR) and the findings agreed with Kawaji et al. [16];
Materials- Elhassan M. A. Saeed, Mohamed A. I. Hamid
Sonawane and Tripathi [17]. This study indicated that the
Data collection - Mohamed A. I. Hamid
RT-PCR is more rapid, specific and sensitive test for
Data analysis- Elhassan M. A. Saeed, Mohamed A. I.
screening and diagnosis of Mycobacterium avium subsp.
Hamid
paratuberculosis in fecal samples of small ruminants.

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Int. J. Life. Sci. Scienti. Res. eISSN: 2455-1716
Hamid et al., 2018
DOI:10.21276/ijlssr.2018.4.5.9

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